Funded in December, 2004: $100000 for 4 years

Baylor researchers will combine MRI with a cellular imaging tracer in mice to see whether progressive accumulation of brain amyloid, a hallmark of Alzheimer’s disease, is associated with progressively slower movement of electrical signals along nerve axons. If so, this imaging method could be tested as an early diagnostic tool for Alzheimer’s disease.

Amyloid (a protein) builds up in the spaces between brain cells in people who develop Alzheimer’s disease. Prior laboratory studies of animal neurons have shown that electrical signals move along the mouse’s nerve axon progressively more slowly when amyloid is present. The investigators plan to use manganese ion with MRI imaging repeatedly to visualize the movement over time of molecules that carry electrochemical signals along the nerves’ axons, in normal aging mice and in a mouse model of progressive brain amyloid accumulation. The researchers hypothesize that axonal transport slows gradually in normal aging mice, but slows markedly in mice with amyloid accumulation, and, that early signs of slowed axonal transport along nerve axons are associated with metabolic changes produced by amyloid accumulation. Slowed axonal transport, therefore, could be an early warning sign of Alzheimer’s disease.

Significance: If these two hypotheses are correct, the study would provide evidence for testing this imaging technology in humans as a means to diagnose Alzheimer’s disease early, when experimental treatments have the best chance of working.

Manganese ion (Mn2+) is paramagnetic, allowing its detection in spin-lattice (T1) - weighted magnetic resonance imaging (MRI) images. This property makes Mn2+ an extremely important potential tool. Mn2+ enters neurons via calcium (Ca2+) channels and, once inside the cells, is sequestered in the endoplasmic reticulum, subsequently transported along microtubules via fast axonal transport and released at the synaptic cleft. We developed the technique of Manganese Enhanced MRI (MEMRI) neuronal tract tracing to allow the in vivo, trans-synaptic visualization of traced neuronal pathways within the CNS. These tracings can be acquired without harming the animal and the Mn2+ ion washes out of the tissue after several days allowing longitudinal studies on the same animal.

We have successfully produced MEMRI tract tracings in the olfactory system, visual system, striatum, and amygdala in mice and in the frontal cortex of rhesus macaques. Because Mn2+ is transported along microtubules via fast axonal transport, it is possible to utilize MRI to dynamically measure the rates of Mn2+ transport, reflective of fast axonal transport rates within the same animal before and during disease progression. We will assess changes in axonal transport in a mouse model of Alzheimer's disease that expresses mutant presenilin (PS1M146V mice,) as well as overexpresses amyloid precursor protein (APP), and will explore the mechanism of any changes.

Robia Pautler, Ph.D.

Hypothesis:Manganese ion (Mn2+) is paramagnetic, allowing its detection in spin-lattice (T1) -weighted magnetic resonance imaging (MRI) images. This property makes Mn2+ an extremely important potential tool. Mn2+ enters neurons via calcium (Ca2+) channels and, once inside the cells, is sequestered in the endoplasmic reticulum, subsequently transported along microtubules via fast axonal transport and released at the synaptic cleft. We developed the technique of Manganese Enhanced MRI (MEMRI) neuronal tract tracing to allow the in vivo, trans-synaptic visualization of traced neuronal pathways within the CNS. These tracings can be acquired without harming the animal, and the Mn2+ ion washes out of the tissue after several days allowing longitudinal studies on the same animal. We have successfully produced MEMRI tract tracings in the olfactory system, visual system, striatum, and amygdala in mice and in the frontal cortex of rhesus macaques.

Axonal transport deficits have been observed in flies and cultured rodent neurons exposed to excess amyloid precursor protein (APP) or amyloid-beta (Ab), but neither the molecular basis of the transport deficit nor the temporal relationship of the transport deficit and the acquisition of Alzheimer's Disease (AD) are known. Additionally, Presenilin 1 is an important gene in the acquisition of Alzheimer's Disease as it is thought to be involved in the development of early Alzheimer's Disease. Mice expressing mutant presenilin (PS1M146V mice) accumulate Ab and develop plaques at an accelerated rate.

Because Mn2+ is transported along microtubules via fast axonal transport, it is possible to utilize MRI to dynamically measure the rates of Mn2+ transport, reflective of fast axonal transport rates within the same animal before and during disease progression.

Because we will be examining structures connected via fiber tracts, measurement of the changes in signal intensity due to Mn2+ accumulation are reflective of the rate that the Mn2+ is being transported. Our group, Nikos Logothetis' group, and Annemie Van der Linden's group have all reported the rate of Mn2+ transport at ~ 2mm/hour. Assuming no partial volume or spin-spin relaxation (T2) effects, the local concentration of Mn2+ will be proportional to the shortening of the spin-lattice (T1) relaxation rate in a specified ROI. Although the spin echo intensity has an exponential dependence on 1/T1, we are assuming that the change in concentration of the Mn2+ ion covers a small enough portion of this spread such that it will appear linear. Therefore, any changes in signal intensity that we observe should be reflective of axonal transport of Mn2+ from the injected structure to the directly connected structure.

Our hypotheses are: 1) Normal aging mice will exhibit declines in axonal transport rates throughout the brain as aging ensues; 2) The presence of excess APP and mutant presenilin 1 causes a reduction in axonal transport prior to the formation of neuritic plaques; and 3) Impairment in the sequestering of Mn2+ into the endoplasmic reticulum results in the observed early decline in axonal transport rates.

Goals:The overall goals of this application are: 1) to determine if a decline in in vivo axonal transport rates in neurons residing in the central nervous system (CNS) occurs in normal aging mice; 2) to determine if transgenic mice overexpressing the familial AD (FAD) mutant of amyloid precursor protein (APP) as well as mutant presenilin 1 (PS1M146V mice) exhibit a significant difference in in vivo axonal transport rates from the observed decline in age-matched controls during the progression of plaque formation; 3) to determine the mechanism of the decline in axonal transport by identifying which steps in Mn2+ uptake or transport are affected.

Methods:The basic format of the experimental procedure is as follows: 1) Administration of the Mn2+ to the targeted areas of the CNS; 2) MRI imaging will occur 1 hour after Mn2+ administration of the targeted area and will last for one hour and twenty minutes, totaling approximately 40 images per dynamic data set. Each image takes approximately 2 minutes to acquire. An n = 5 control mice and n = 5 for the AD mice will be collected for each age point. All mice will be age and sex matched. The zero time point for imaging will be one hour after nasal lavage; 3) Analysis of the MRI data to ascertain the rate of transport of the Mn2+ ion.

MRI Imaging: T1-weighted, spin-echo 2D data sets will be acquired of the mouse brain using a horizontal bore 9.4T Bruker Avance imaging spectrometer with a micro-imaging gradient insert and a 35 mm volume coil (Bruker Instruments,Billerica, MA). The imaging parameters are as follows: Multi-Slice/Multi Echo 2D imaging protocol, matrix dimensions = 128x128; FOV = 1 cm x 1; slice thickness = 1 mm, resulting in an in-plane spatial resolution of 78 mm. The repetition time (TR) = 504.1ms; echo time (TE) = 8.2 ms and NA = 2. The total imaging time per image is 2 minutes. A total of 40 image sets will be collected. The short TR ensures a heavily T1-weighted image that will provide positive signal enhancement in regions with an accumulation of the paramagnetic Mn2+. Because axonal transport is a temperature dependent process, it is very important to monitor and maintain the body temperature of the mouse. The body temperature of the mouse will be monitored and maintained at 36.90 C utilizing a heated, circulating water blanket.

Lay Results:
Alzheimer’s disease (AD) is an age-related, neurodegenerative disease that is one of the leading causes of dementia, afflicting 1% of people under the age of 60 to more than 40% of people over the age of 85. The symptoms of this disease are typified by memory loss and a progressive decline of cognitive abilities. The goal of this project was to utilize in vivo MRI imaging to measure changes in brain physiology. Specifically, we measured if there are changes in the rates of transport in vital biological materials in the brain. Our data indicate that there are large deficits in these important transport processes that occur very early on in AD progression and much earlier before plaque formation occurs. We are currently using this MRI technique to evaluate therapeutic strategies to improve this transport deficit in the hopes that we can use this technology as a means to screen drugs treatments as well as understand more about AD.

Scientific Results:
Alzheimer’s disease (AD) is an age-related, neurodegenerative disease that is one of the leading causes of dementia, afflicting 1% of people under the age of 60 to more than 40% of people over the age of 85. The symptoms of this disease are typified by memory loss and a progressive decline of cognitive abilities. This disease is characterized by the extracellular deposition of amyloid-beta (Ab) aggregates known as plaques that are surrounded by dystrophic neurites and activated glial cells as well as intracellular neurofibrillary tangles that are comprised of hyperphosphorylated tau protein aggregates. As detailed in the February 2006 Alzheimer’s Research Forum, one of the most critical needs in AD research is the identification of biomarkers that can not only predict disease but also monitor responses to treatment. In this project, we first propose to utilize a novel MRI methodology, Manganese Enhanced MRI (MEMRI) that we helped develop to delineate the role of Ab on in vivo axonal transport rates in mouse models of AD. Our data indicate that in vivo axonal transport rates decrease during Ab accumulation and prior to plaque formation. We are evaluating pharmacological and genetic strategies to improve these observed axonal transport deficits.

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